Optical Observation of DNA Translocation Dynamics in SiN Nanopores

Abstract

Nanopore-based technologies have shown a great promise for developing single-molecule DNA sequencing. In rough classification, two types of measurement methods are employed to detect DNA translocation through the nanopore; ionic current detection and optical detection. The optical detection enables to visualize the whole process of translocation, while the ionic current method detects only the current blocking time and amplitude. In this study, we demonstrated the salt dependence of single-molecule DNA translocation process through SiN nanopores by a fluorescence microscopic measurement. In particular, we investigated the deformation of DNA coil structure immediately before and after translocation. We fabricated a 100 nm diameter nanopore by milling a 20 nm thickness SiN membrane by focused ion beam. A blue laser diode was used to excite YOYO-1, which stains 10 kbp DNA sample. The confocal microscope setup allows to observe the DNA dynamics in the vicinity of nanopore with a small observation volume (200 nm in lateral diameter). The time trace of fluorescence signal visualizes the change in volume fraction of DNA molecule inside the observation volume. The rising time and the decay time of the signal represent the capture-translocation process and the drift-diffusion (escape) process, respectively. We found that the rising time increased with the salt concentration. This might be attributed to the fact that the binding of counterion to negatively charged DNA molecule causes the change in its electrophoretic mobility and conformation. Due to the decrease in persistence length and hence stiffness with the salt concentration, the DNA molecule remains less stretched for a longer time before translocation. The explanation is consistent with the result that the rising time decreases with the applied voltage. The higher electric field makes the DNA molecule to be more stretched and more easily captured by the nanopore.